Staphylococcal protein A binding to canine IgG and IgM.
Staphylococcal protein A (SpA) binds with high affinity to immunoglobulins from many species, making it a useful reagent for immunoassays and immunoglobulin purification procedures. However, its use is limited by poor reactivity with some immunoglobulin subclasses including human IgG3 and murine IgG1. Reports of SpA's reactivity with canine immunoglobulins have been inconsistent. Because the most recent reports indicated a much greater reactivity of purified SpA with canine immunoglobulins than was suggested by our observations, we quantitatively reevaluated the binding of canine IgG and IgM to Cowan I strain SpA under the conditions of use in our laboratory. IgG and IgM were purified from pooled normal canine plasma by affinity chromatography with heavy chain specific polyclonal anti-IgG and anti-IgM antibodies. The purified IgG and IgM were assessed for SpA reactivity by affinity chromatography using a SpA-agarose column. The relative proportions of total chromatographed IgG or IgM in the flow-through (SpA-nonbindable) and eluate (SpA-bindable) fractions were determined by absorbance at 280 nm. The IgG and IgM in each immunoglobulin fraction were also nonspecifically adsorbed to microtitration plates and tested for reactivity with 125I-SpA using a solid phase immunoradiometric assay (IRMA). Approximately 18% of the affinity purified canine IgG and 33% of the affinity purified canine IgM did not bind to the SpA affinity column and were also unreactive with 125I-SpA using the IRMA. A second approach using a different polyclonal antibody to canine IgG yielded similar results: about 21% of the purified IgG was unreactive with SpA. Incomplete reactivity of SpA with canine IgG and IgM limits the usefulness of SpA in canine immunologic procedures.
Scott
M A
MA
Department of Pathology, Michigan State University, East Lansing, USA. scott@vmdl.missouri.edu
Davis
J M
JM
Schwartz
K A
KA
D000818:Animals; D002846:Chromatography, Affinity; D004285:Dogs; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007075:Immunoglobulin M / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000378:metabolism*
D007074:Immunoglobulin G; D007075:Immunoglobulin M; D013205:Staphylococcal Protein A